CN114958897A - 可高效表达饲用低温角蛋白酶的枯草芽孢杆菌构建方法 - Google Patents
可高效表达饲用低温角蛋白酶的枯草芽孢杆菌构建方法 Download PDFInfo
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- CN114958897A CN114958897A CN202210685139.9A CN202210685139A CN114958897A CN 114958897 A CN114958897 A CN 114958897A CN 202210685139 A CN202210685139 A CN 202210685139A CN 114958897 A CN114958897 A CN 114958897A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
本发明公开了一种促表达元件、重组载体、菌株，促表达元件由p43启动子、hpall启动子、phod信号肽组成，还可以包括apre启动子；所述重组表达载体由phy‑p43载体、hpall启动子、Ker基因和phod信号肽组成，还包括apre启动子；以所述表达元件或重组表达载体构建菌株，并以该菌株发酵产角蛋白酶，显著提升了酶的活力。
Description
技术领域
本发明涉及基因工程技术领域，更具地说是涉及一种促表达元件、重组表达载体、菌株及应用。
背景技术
角蛋白酶是一种特殊碱性丝氨酸蛋白酶，包含二硫键水解酶和多肽水解酶，可高效打开二硫键，降解角蛋白、醇溶蛋白、多肽和其他蛋白质。角蛋白酶耐酸性好，过胃能力强，在肠道发挥高效酶活，耐热性良好，在颗粒料的生产中具有一定的优势，研究表明，角蛋白酶在不同类型的日粮均可发挥作用。玉米-豆粕型基础日粮中添加角蛋白酶可提高肉鸡日增重、饲料利用率以及脂肪消化率。小麦-豆粕型基础日粮中添加角蛋白酶可提高淀粉、脂肪、能量表观利用率以及氨基酸利用率。角蛋白酶可全方位、高效水解饲料中蛋白质，在提升畜禽对蛋白质的消化利用率方面具有独特优势。据文献报道，Brevibacillus brevisUS575角蛋白酶最适温度为40℃，与动物肠道环境相似，有在饲料中应用的潜力。
枯草芽孢杆菌是重要的工业酶蛋白表达系统之一，具有分泌表达、无致病性、发酵技术成熟等优势，在酶制剂生产上得到广泛应用。枯草芽孢杆菌是蛋白酶和淀粉酶主要的生产菌株。
启动子是枯草芽孢杆菌高效表达目的蛋白的关键元件之一，启动子的转录强度严重影响着外源蛋白的表达量，启动子分为诱导型、时期特异型、组成型和自诱导型。组成型启动子是一类能够持续表达目的蛋白，且在发酵生产过程中不需要添加任何诱导物的启动子。不同的外源蛋白对启动子的适应性不同，因此，改造启动子以实现目的基因的高效表达是目前研究的热点之一。
因此，如何通过改造启动子来构建表达元件、重组表达载体，进而构建能够产角蛋白酶的菌株是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种重组表达载体，并将载体转化至枯草芽孢杆菌菌株中，产饲用角蛋白酶，显著提高了酶活。
为了实现上述目的，本发明采用如下技术方案：
一种促表达元件，所述促表达元件由p43启动子、hpall启动子和phod信号肽组成。
作为与上述技术方案相同的发明构思，本发明还请求保护一种促表达元件，所述促表达元件由p43启动子、hpall启动子、apre启动子和phod信号肽组成。
作为与上述技术方案相同的发明构思，本发明还请求保护一种重组表达载体，所述重组表达载体由phy-p43载体、hpall启动子、Ker基因和phod信号肽组成。
作为与上述技术方案相同的发明构思，本发明还请求保护一种重组表达载体，所述重组表达载体由phy-p43载体、hpall启动子、apre启动子、Ker基因和phod信号肽组成。
作为与上述技术方案相同的发明构思，本发明还请求保护一种工程菌株，所述工程菌株任一所述的促表达元件为表达元件。
作为与上述技术方案相同的发明构思，本发明还请求保护一种工程菌株，所述工程菌株以任一所述的重组表达载体进行表达。
作为与上述技术方案相同的发明构思，本发明还请求保护所述的促表达元件在发酵产角蛋白酶中的应用。
作为与上述技术方案相同的发明构思，本发明还请求保护任一所述的重组表达载体在发酵产角蛋白酶中的应用。
作为与上述技术方案相同的发明构思，本发明还请求保护任一所述的工程菌株在发酵产角蛋白酶中的应用。
经由上述的技术方案可知，与现有技术相比，本发明采用多启动子串联策略提高角蛋白酶在枯草芽孢杆菌中的分泌表达水平。相比于单p43启动子的工程菌发酵上清液中的角蛋白酶酶活为的3700U/mL，p43与hpall启动子串联的发酵上清液中的角蛋白酶酶活为4950U/mL，p43与hpall和apre启动子串联后，发酵上清液中的角蛋白酶酶活为6780U/mL，表明多启动子串联策略对于角蛋白酶分泌量有很大提高。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为本发明phy-p43-phod-Ker载体图谱。
图2附图为本发明phy-p43-Phpall-phod-Ker载体图谱。
图3附图为本发明phy-p43-Phpall-Papre-phod-Ker载体图谱。
图4附图为启动子串联顺序图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
根据NCBI处获得的Bacillus pumilus角蛋白酶氨基酸序列(GenBankACM47735.1)SEQ ID NO.15经枯草芽孢杆菌密码子优化得到SEQ ID NO.1所示的基因序列，启动子HpaII基因序列SEQ ID NO.2，启动子apre基因序列SEQ ID NO.3，phod信号肽基因序列SEQ ID NO.4均由擎科生物分别进行全基因合成，连接在pUC57克隆载体上。
实施例1多启动子串联表达载体的构建
标准的PCR反应体系如下：总体积为50μL的PCR反应体系中，加入25μL 2xTaq PlusPCR Master Mix，2.5μL(10μM)上游引物PF，2.5μL(10μM)下游引物PR，1μL模板，加灭菌蒸馏水至50μL；
PCR反应程序：(1)94℃预变性3min，(2)94℃变性30sec，(3)55℃退火30sec，(4)72℃延伸(1kb延伸1min)，步骤(2)～(4)共进行30个循环，4℃保存PCR产物。
PCR反应结束后，进行琼脂糖凝胶电泳检验目的基因大小是否正确，结果正确后，进行切胶回收，用天根生化科技有限公司生产的凝胶回收试剂盒进行目的基因回收(过程按说明书进行)。
标准的酶切酶连体系按限制性核酸内切酶说明书进行，首先将目的基因进行酶切，然后将目标载体进行酶切，胶回收后按酶连体系进行酶连反应，然后转入大肠杆菌DH5α感受态细胞中，抗性平板筛选，将长出的阳性克隆进行PCR验证是否连接成功。
phy-p43-phod-Ker表达载体构建
以合成的基因序列为模版，采用标准的PCR扩增体系，以Ker-F和Ker-R为引物，扩增角蛋白酶目的基因Ker，在5’端引入BamHI酶切位点，3’端引入EcoRI酶切位点，使用标准的酶切酶连体系，构phy-p43-Ker表达载体。接着以phod-F和phod-R为引物，以标准PCR扩增体系扩增phod信号肽部分，在5’端引入BglII酶切位点，3’端引入SalI酶切位点，使用标准的酶切酶连体系，构建phy-p43-phod-Ker表达载体。
Ker-F：5’-cgggatccATGTGCGTTAAAAAAAAAAACGTTATG-3’；SEQ ID NO.5
Ker-R：5’-cggaattc TTAGTTAGAAGCAGCTTGAACGTTG-3’；SEQ ID NO.6
Phod-F：5’-gaagatctATGGCATACGACAGTCGT-3’；SEQ ID NO.7
Phod-R：5’-gcgtcgacTACTTCAAAGGCCCCAAC-3’；SEQ ID NO.8
phy-p43-Phpall-phod-Ker表达载体构建
以合成的基因序列为模版，采用标准的PCR扩增体系，以hpall-F和hpall-R为引物，扩增hpall promoter部分，在5’端引入BglII酶切位点，3’端引入RBS序列，接着以phod-F1和phod-R为引物，扩增phod信号肽部分，在5’端引入RBS序列，3’端引入SalI酶切位点，hpall-F和phod-R为引物，通过RBS序列作为重叠区域进行融合PCR，连接Phpall和phod信号肽部分，连接成功后，使用标准的酶切酶连体系，构建phy-p43-Phpall-phod-Ker表达载体。
Hpall-F：5’-gaagatctTACTACCTGTCCCTTGCTGAT-3’；SEQ ID NO.9
Hpall-R：5’-gtgtacattcctctcttATGTAAATCGCTCCTTTTTAGGT-3’；SEQ ID NO.10
Phod-F1：5’-aagagaggaatgtacACATGGCATACGACAGTCGT-3’；SEQ ID NO.11
Phod-R：5’-gcgtcgacTACTTCAAAGGCCCCAAC-3’；SEQ ID NO.12
phy-p43-Phpall-Papre-phod-Ker表达载体构建
以合成的基因序列为模版，采用标准的PCR扩增体系，以Papre-F和Papre-R为引物，扩增apre promoter部分，在5’端和3’端分别引入RBS序列，首先，以hpall-F和apre-R通过RBS序列作为重叠区域进行融合PCR，连接Phpall和Papre部分，构建Phpall-Papre表达盒，然后，继续通过融合PCR，以hpall-F和phod-R构建Phpall-Papre-phod表达盒，使用标准的酶切酶连体系，构建phy-p43-Phpall-Papre-phod-Ker表达载体。
apre-F：5’-aagagaggaatgtacacGTTCTTTCTGTATGAAAATAGTT-3’；SEQ ID NO.13
apre-R：5’-gtgtacattcctctcttAGCCTGCGCAGACATGTTG-3’；SEQ ID NO.14
实施例2枯草芽孢杆菌工程菌的构造
1、枯草芽孢杆菌感受态细胞的制备
(1)将枯草芽孢杆菌WB600甘油菌在LB培养基中划线培养，37℃培养过夜；然后将长出来的单菌落接到新鲜的LB液体培养基中，37℃下激烈震荡培养至OD600＝0.4-0.6。
(2)取1ml培养液转移到无菌离心管中，在冰上放置30min后在4℃，4000rpm条件下离心5min，弃上清，回收菌体。
(3)用1ml预冷的无菌去离子水清洗菌体，4℃,4000rpm条件下离心5min,弃上清，重复2次。
(4)用1mLHG溶液再清洗菌体，4℃,4000rpm离心5min，弃上清，用200μL的HG溶液重悬菌体,即可用于电激或置于-20℃保存。
HG溶液:10％甘油，1mM Hepes(pH7.0)。
2、枯草芽孢杆菌感受态细胞的电转化和筛选
(1)将待转化的重组表达质粒加入制备好的枯草芽孢杆菌感受态细胞中，4℃保温10min。
(2)在2.5KV/cm,25uF,720Q条件下电击6ms进行电转化,然后4℃保温10min。
(3)加入500μLSOC培养基，于37℃、100rpm条件下复苏培养2h,取200ul涂布于LB(含筛选所需的抗生素)固体培养基上，将平板置于室温直至液体被吸收，倒置培养皿，于37℃培养12-16h。
(4)挑选转化子，提质粒验证是否转化成功。
SOC液体培养基:蛋白陈2％、酵母提取物0.5％、NaCl 0.05％、KCl 2.5mM、MgCl210mM、葡萄糖20mM，pH7.0。
实施例3不同启动子对枯草芽孢杆菌产角蛋白酶影响
种子培养基：酵母粉5g/L，蛋白胨10g/L。
发酵培养基:蛋白陈20g/L、酵母粉10g/L、蔗糖20g/L、KH2PO4
3g/L、Na2HPO46g/L、MgSO40.3g/L。
将重组质粒phy-p43-Phpall-phod-Ker、phy-p43-Phpall-Papre-phod-Ker成功转化的枯草芽孢工程菌接入种子培养基进行发酵，获得种子发酵液，然后进行摇瓶发酵：向500mL三角瓶中装入250mL液体发酵培养基，然后将种子培养液接种与发酵培养基，控制OD600为0.5，转速200rpm，37℃，发酵培养48小时，得到生产发酵的菌液。
水溶性角蛋白(购自梯希爱(上海)化成工业发展有限公司，产品编码:KO043)；
角蛋白酶酶活的测定:取50μL适当稀释的发酵上清液,加入150μL50mM的Gly/NaOH溶液作为缓冲液和100μL浓度为2.5％的水溶性角蛋白作为底物,混匀后于40℃下反应20min；加入200μL4％(w/v)的三氯乙酸(TCA)终止反应，室温8000rpm离心3min。取上清200uL,加入1mL 4％(w/v)的Na2CO3和200uL的福林酚试剂,混匀后50℃下显色10min,使用0.5cm石英比色皿于660nm下测定清液吸光值；实验组3个平行,空白对照是在加入底物之前先加入反应终止剂TCA,其余操作同上。
酶活的定义:在该条件下OD660每升高0.001所需酶量为一个酶活力单位(1U)。
酶活力检测结果如下：WB600-phy-p43-phod-Ker的发酵上清液中的角蛋白酶酶活为3700U/mL，WB600-phy-p43-Phpall-phod-Ker的发酵上清液中的角蛋白酶酶活为4950U/mL，WB600-phy-p43-Phpall-Papre-phod-Ker的发酵上清液中的角蛋白酶酶活为6780U/mL，结果表明：多启动子串联的重组质粒较单启动子而言酶活有显著的提升。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。对于实施例公开的装置而言，由于其与实施例公开的方法相对应，所以描述的比较简单，相关之处参见方法部分说明即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
<110> 中农华威生物制药（湖北）有限公司
<120> 一种可高效表达饲用低温角蛋白酶的枯草芽孢杆菌构建
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1149
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgtgcgtta aaaaaaaaaa cgttatgaca tctgttcttc ttgctgttcc tcttcttttc 60
tctgctggct tcggcggcac aatggctaac gctgaaacag tttctaaaac agattctgaa 120
aaatcttaca tcgttggctt caaagcttct gctacaacaa actcttctaa aaaacaagct 180
gttatccaaa acggcggcaa acttgaaaaa caataccgtc ttatcaacgc tgctcaagtt 240
aaaatgtctg aacaagctgc taaaaaactt gaacatgatc cttctatcgc ttacgttgaa 300
gaagatcata aagctgaagc ttacgctcaa acagttcctt acggcatccc tcaaatcaaa 360
gctcctgctg ttcatgctca aggctacaaa ggcgctaacg ttaaagttgc tgttcttgat 420
acaggcatcc atgctgctca tcctgatctt aacgttgctg gcggcgcttc tttcgttcct 480
tctgaaccta acgctacaca agatttccaa tctcatggca cacatgttgc tggcacaatc 540
gctgctcttg ataacacaat cggcgttctt ggcgttgctc ctaacgcttc tctttacgct 600
gttaaagttc ttgatcgtaa cggcgatggc caatactctt ggatcatctc tggcatcgaa 660
tgggctgttg ctaacaacat ggatgttatc aacatgtctc ttggcggccc ttctggctct 720
acagctctta aaaacgctgt tgatacagct aacaaccgtg gcgttgttgt tgttgctgct 780
gctggcaact ctggctcttc tggctctcgt tctacagttg gctaccctgc taaatacgat 840
tctacaatcg ctgttgctaa cgttaactct aacaacgttc gtaactcttc ttcttctgct 900
ggccctgaac ttgatgtttc tgctcctggc acatctatcc tttctacagt tccttcttct 960
ggctacacat cttacacagg cacatctatg gcttctcctc atgttgctgg cgctgctgct 1020
cttatccttt ctaaaaaccc taaccttaca aactctcaag ttcgtcaacg tcttgaaaac 1080
acagctacac ctcttggcga ttctttctac tacggcaaag gccttatcaa cgttcaagct 1140
gcttctaac 1149
<210> 2
<211> 272
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tactacctgt cccttgctga tttttaaacg agcacgagag caaaaccccc ctttgctgag 60
gtggcagagg gcaggttttt ttgtttcttt tttctcgtaa aaaaaagaaa ggtcttaaag 120
gttttatggt tttggtcggc actgccgaca gcctcgcaga gcacacactt tatgaatata 180
aagtatagtg tgttatactt tacttggaag tggttgccgg aaagagcgaa aatgcctcac 240
atttgtgcca cctaaaaagg agcgatttac at 272
<210> 3
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gttctttctg tatgaaaata gttatttcga gtctctacgg aaatagcgag agatgatata 60
cctaaataga gataaaatca tctcaaaaaa atgggtctac taaaatatta ttccatctat 120
tacaataaat tcacagaata gtctttaagt aagtctactc tgaacttaag caaaaggaga 180
ggggtgagaa gcaaaaaatt gtggatcagc ttgttgtttg cgttaacgtt aatctttacg 240
atggcgttca gcaacatgtc tgcgcaggct 270
<210> 4
<211> 162
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggcatacg acagtcgttt tgatgaatgg gtacagaaac tgaaagagga aagctttcaa 60
aacaatacgt ttgaccgccg caaatttatt caaggagcgg ggaagattgc aggactttct 120
cttggattaa cgattgccca gtcggttggg gcctttgaag ta 162
<210> 5
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgggatccat gtgcgttaaa aaaaaaaacg ttatg 35
<210> 6
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cggaattctt agttagaagc agcttgaacg ttg 33
<210> 7
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gaagatctat ggcatacgac agtcgt 26
<210> 8
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gcgtcgacta cttcaaaggc cccaac 26
<210> 9
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gaagatctta ctacctgtcc cttgctgat 29
<210> 10
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gtgtacattc ctctcttatg taaatcgctc ctttttaggt 40
<210> 11
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aagagaggaa tgtacacatg gcatacgaca gtcgt 35
<210> 12
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gcgtcgacta cttcaaaggc cccaac 26
<210> 13
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
aagagaggaa tgtacacgtt ctttctgtat gaaaatagtt 40
<210> 14
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gtgtacattc ctctcttagc ctgcgcagac atgttg 36
<210> 15
<211> 383
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Cys Val Lys Lys Lys Asn Val Met Thr Ser Val Leu Leu Ala Val
1 5 10 15
Pro Leu Leu Phe Ser Ala Gly Phe Gly Gly Thr Met Ala Asn Ala Glu
20 25 30
Thr Val Ser Lys Thr Asp Ser Glu Lys Ser Tyr Ile Val Gly Phe Lys
35 40 45
Ala Ser Ala Thr Thr Asn Ser Ser Lys Lys Gln Ala Val Ile Gln Asn
50 55 60
Gly Gly Lys Leu Glu Lys Gln Tyr Arg Leu Ile Asn Ala Ala Gln Val
65 70 75 80
Lys Met Ser Glu Gln Ala Ala Lys Lys Leu Glu His Asp Pro Ser Ile
85 90 95
Ala Tyr Val Glu Glu Asp His Lys Ala Glu Ala Tyr Ala Gln Thr Val
100 105 110
Pro Tyr Gly Ile Pro Gln Ile Lys Ala Pro Ala Val His Ala Gln Gly
115 120 125
Tyr Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile His
130 135 140
Ala Ala His Pro Asp Leu Asn Val Ala Gly Gly Ala Ser Phe Val Pro
145 150 155 160
Ser Glu Pro Asn Ala Thr Gln Asp Phe Gln Ser His Gly Thr His Val
165 170 175
Ala Gly Thr Ile Ala Ala Leu Asp Asn Thr Ile Gly Val Leu Gly Val
180 185 190
Ala Pro Asn Ala Ser Leu Tyr Ala Val Lys Val Leu Asp Arg Asn Gly
195 200 205
Asp Gly Gln Tyr Ser Trp Ile Ile Ser Gly Ile Glu Trp Ala Val Ala
210 215 220
Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser
225 230 235 240
Thr Ala Leu Lys Asn Ala Val Asp Thr Ala Asn Asn Arg Gly Val Val
245 250 255
Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Ser Arg Ser Thr
260 265 270
Val Gly Tyr Pro Ala Lys Tyr Asp Ser Thr Ile Ala Val Ala Asn Val
275 280 285
Asn Ser Asn Asn Val Arg Asn Ser Ser Ser Ser Ala Gly Pro Glu Leu
290 295 300
Asp Val Ser Ala Pro Gly Thr Ser Ile Leu Ser Thr Val Pro Ser Ser
305 310 315 320
Gly Tyr Thr Ser Tyr Thr Gly Thr Ser Met Ala Ser Pro His Val Ala
325 330 335
Gly Ala Ala Ala Leu Ile Leu Ser Lys Asn Pro Asn Leu Thr Asn Ser
340 345 350
Gln Val Arg Gln Arg Leu Glu Asn Thr Ala Thr Pro Leu Gly Asp Ser
355 360 365
Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ser Asn
370 375 380
Claims (9)
1.一种促表达元件，其特征在于，所述促表达元件由p43启动子、hpall启动子和phod信号肽组成。
2.一种促表达元件，其特征在于，所述促表达元件由p43启动子、hpall启动子、apre启动子和phod信号肽组成。
3.一种重组表达载体，其特征在于，所述重组表达载体由phy-p43载体、hpall启动子、Ker基因和phod信号肽组成。
4.一种重组表达载体，其特征在于，所述重组表达载体由phy-p43载体、hpall启动子、apre启动子、Ker基因和phod信号肽组成。
5.一种工程菌株，其特征在于，所述工程菌株以权利要求1-2任一所述的促表达元件为表达元件。
6.一种工程菌株，其特征在于，所述工程菌株以权利要求3-4任一所述的重组表达载体进行表达。
7.权利要求1-2任一所述的的促表达元件在发酵产角蛋白酶中的应用。
8.权利要求3-4任一所述的重组表达载体在发酵产角蛋白酶中的应用。
9.权利要求5-6任一所述的工程菌株在发酵产角蛋白酶中的应用。
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Cited By (2)
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| CN114875057A (zh) * | 2022-06-14 | 2022-08-09 | 中农华威生物制药(湖北)有限公司 | 一种可高效表达饲用低温酸性α-淀粉酶的枯草芽孢杆菌的构建方法 |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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| CN114875057A (zh) * | 2022-06-14 | 2022-08-09 | 中农华威生物制药(湖北)有限公司 | 一种可高效表达饲用低温酸性α-淀粉酶的枯草芽孢杆菌的构建方法 |
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